Assembly, activation and anti-CRISPR inhibition of a type I-C cascade




O'Brien, Roisin Esther

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Type I CRISPR-Cas systems employ multi-subunit Cascade effector complexes to target foreign nucleic acids for destruction. The type I-C subtype contains a minimal Cascade effector complex, encoded by only three unique genes in its operon. Despite being one of the most common subtypes in nature, relatively little information exists about its effector complex. As the type I-C CRISPR system emerges as a popular tool for genome engineering, it is crucial to understand the mechanisms behind type I-C interference and provide the structural blueprint required for rational Cascade re-engineering. Here, we present structures of D. vulgaris type I-C Cascade at various stages of target interference, revealing mechanisms that underpin assembly, allosteric activation, and anti-CRISPR inhibition. Characterizing the assembly of the type I-C Cascade through both structural and analytical approaches, uncovers previously overlooked, non-canonical small subunits (Chapter 1). Structures of type I-C Cascade at various stages of target capture reveal novel mechanisms of PAM recognition and NTS stabilization (Chapter 2). And finally, structural insights into how two distinct anti-CRISPR proteins utilize distinct strategies highlight a shared mechanism of type I-C Cascade inhibition by blocking PAM scanning (Chapter 3).




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